The effects of a monoclonal antibody on the imipramine (IMI) metabolism by rat liver microsomes are examined. Two primary metabolites of IMI, hydroxyimipramine (OHIMI) and desmethylimipramine (DIMI) were measured by HPLC. Lineweaver-Burk plots were obtained for the two metabolites with microsomes from control rats and rats treated with phenobarbital (PB) or Beta-naphthoflavone (BNF). Moreover, the Km values calculated from these plots were nearly identical for both of the products regardless of the source of the microsomes and the ratio of the Vmax values for the control and PB-treated rats were similar. Thus the kinetic parameters alone could not differentiate between the isozymes of cytochrome P-450 that metabolize imipramine. Metabolism of IMI by liver microsomes from PB-treated rats was inhibited by an anti-cytochrome P-450 PB-B monoclonal antibody (anti PB-B-M Ab) in a dose dependent way with maximum 50% inhibition. The titration curves for the inhibition of OHIMI and DMI production cannot be dissociated, indicating nearly identical antigenic sites. Although the Lineweaver-Burk plot for the residual activities remained straight for N-demethylation, the curve for hydroxylation was broken. Thus despite the similarity in kinetic parameters, the two reactions are done by at least two isozymes in microsomes from PB-treated rats. The ratio of both metabolites OHIMI/DIMI was 1.3/5.7 = 0.228 in the phenobarbital induced microsomal system, but the ratio was 1.33/ 0.628 = 2.118 in the reconstituted system using purified cytochrome P-450 PB-B. These results suggest either the isolateld enzyme is not the major form in microsomes that metabolizes IMI or that the reconstituted system does not provide accurate kinetic information.